Guinea-pig megakaryocytes can respond to external ADP by activating Ca2(+)-dependent potassium conductance

Abstract

1. The responses of megakaryocytes to adenosine diphosphate (ADP) were studied using whole-cell patch electrodes and a Ca2(+)-sensitive fluorescent dye, Fura-2. Megakaryocytes (diameter, 17-42 microns) were mechanically dissociated from the bone marrow of adult guinea-pigs and ADP (1-10 microM) was pressure-applied to megakaryocytes under recording. 2. In megakaryocytes immersed in standard saline, ADP evoked an obvious outward current at a membrane potential of -63 mV. The current was identified as a K(+)-carried current, since the reversal potential depended distinctly on the external K+ concentration, but it showed no changes after removal of external Na+. The amplitude of evoked K+ currents showed considerable intercell variation, which is presumably due to differences of current density in the membrane. 3. During application of ADP, the evoked K+ current was not sustained but slowly decayed to become negligible within 10-20 s, suggesting the appearance of desensitization. The response of the megakaryocyte to ADP recovered slowly and returned to an original level after 4-5 min of continuous washing. 4. When the intracellular free Ca2+ concentration ([Ca2+]i) was measured using the Ca2(+)-sensitive fluorescent dye, Fura-2, application of 10 microM-ADP induced an increase of [Ca2+]i by about 5-fold, which was followed by a gradual decay to the original level within 30-50 s. Roles of internal Ca2+ for activating the K+ current were confirmed by observing (1) enhancement of evoked currents by the use of internal saline containing no Ca2+ chelators and (2) generation of prolonged K+ current by application of a Ca2+ ionophore, A23187, to the megakaryocyte. 5. In a fraction of the megakaryocytes, spontaneous hyperpolarization of the resting membrane potential was observed. The hyperpolarization seemed to result from the activation of K+ channels in the membrane, which was caused by spontaneous release of Ca2+ from the internal storage site. 6. It was concluded that megakaryocytes of the guinea-pig can respond to external ADP by increasing [Ca2+]i and consequently by activating Ca2(+)-dependent K+ channels in the membrane.